In Vitro Uptake of 140 kDa Bacillus thuringiensis Nematicidal Crystal Proteins by the Second Stage Juvenile of Meloidogyne hapla

Plant-parasitic nematodes (PPNs) are piercing/sucking pests, which cause severe damage to crops worldwide, and are difficult to control. The cyst and root-knot nematodes (RKN) are sedentary endoparasites that develop specialized multinucleate feeding structures from the plant cells called syncytia or giant cells respectively. Within these structures the nematodes produce feeding tubes, which act as molecular sieves with exclusion limits. For example, Heterodera schachtii is reportedly unable to ingest proteins larger than 28 kDa. However, it is unknown yet what is the molecular exclusion limit of the Meloidogyne hapla. Several types of Bacillus thuringiensis crystal proteins showed toxicity to M. hapla. To monitor the entry pathway of crystal proteins into M. hapla, second-stage juveniles (J2) were treated with NHS-rhodamine labeled nematicidal crystal proteins (Cry55Aa, Cry6Aa, and Cry5Ba). Confocal microscopic observation showed that these crystal proteins were initially detected in the stylet and esophageal lumen, and subsequently in the gut. Western blot analysis revealed that these crystal proteins were modified to different molecular sizes after being ingested. The uptake efficiency of the crystal proteins by the M. hapla J2 decreased with increasing of protein molecular mass, based on enzyme-linked immunosorbent assay analysis. Our discovery revealed 140 kDa nematicidal crystal proteins entered M. hapla J2 via the stylet, and it has important implications in designing a transgenic resistance approach to control RKN

Expression Patterns of Genes Involved in Sugar Metabolism and Accumulation during Apple Fruit Development

Both sorbitol and sucrose are imported into apple fruit from leaves. The metabolism of sorbitol and sucrose fuels fruit growth and development, and accumulation of sugars in fruit is central to the edible quality of apple. However, our understanding of the mechanisms controlling sugar metabolism and accumulation in apple remains quite limited. We identified members of various gene families encoding key enzymes or transporters involved in sugar metabolism and accumulation in apple fruit using homology searches and comparison of their expression patterns in different tissues, and analyzed the relationship of their transcripts with enzyme activities and sugar accumulation during fruit development. At the early stage of fruit development, the transcript levels of sorbitol dehydrogenase, cell wall invertase, neutral invertase, sucrose synthase, fructokinase and hexokinase are high, and the resulting high enzyme activities are responsible for the rapid utilization of the imported sorbitol and sucrose for fruit growth, with low levels of sugar accumulation. As the fruit continues to grow due to cell expansion, the transcript levels and activities of these enzymes are down-regulated, with concomitant accumulation of fructose and elevated transcript levels of tonoplast monosaccharide transporters (TMTs), MdTMT1 and MdTMT2; the excess carbon is converted into starch. At the late stage of fruit development, sucrose accumulation is enhanced, consistent with the elevated expression of sucrose-phosphate synthase (SPS), MdSPS5 and MdSPS6, and an increase in its total activity. Our data indicate that sugar metabolism and accumulation in apple fruit is developmentally regulated. This represents a comprehensive analysis of the genes involved in sugar metabolism and accumulation in apple, which will serve as a platform for further studies on the functions of these genes and subsequent manipulation of sugar metabolism and fruit quality traits related to carbohydrates